Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/β-Catenin signaling through Frizzled2-LRP6 assembly

Background Cancer stem-like cells (CSCs) play a pivotal role in lung tumor formation and progression. Nerve injury-induced protein 1 (Ninjurin1, Ninj1) has been implicated in lung cancer; however, the pathological role of Ninj1 in the context of lung tumorigenesis remains largely unknown. Methods The role of Ninj1 in the survival of non-small cell lung cancer (NSCLC) CSCs within microenvironments exhibiting hazardous conditions was assessed by utilizing patient tissues and transgenic mouse models where Ninj1 repression and oncogenic KrasG12D/+ or carcinogen-induced genetic changes were induced in putative pulmonary stem cells (SCs). Additionally, NSCLC cell lines and primary cultures of patient-derived tumors, particularly Ninj1high and Ninj1low subpopulations and those with gain- or loss-of-Ninj1 expression, and also publicly available data were all used to assess the role of Ninj1 in lung tumorigenesis. Results Ninj1 expression is elevated in various human NSCLC cell lines and tumors, and elevated expression of this protein can serve as a biomarker for poor prognosis in patients with NSCLC. Elevated Ninj1 expression in pulmonary SCs with oncogenic changes promotes lung tumor growth in mice. Ninj1high subpopulations within NSCLC cell lines, patient-derived tumors, and NSCLC cells with gain-of-Ninj1 expression exhibited CSC-associated phenotypes and significantly enhanced survival capacities in vitro and in vivo in the presence of various cell death inducers. Mechanistically, Ninj1 forms an assembly with lipoprotein receptor-related protein 6 (LRP6) through its extracellular N-terminal domain and recruits Frizzled2 (FZD2) and various downstream signaling mediators, ultimately resulting in transcriptional upregulation of target genes of the LRP6/β-catenin signaling pathway. Conclusions Ninj1 may act as a driver of lung tumor formation and progression by protecting NSCLC CSCs from hostile microenvironments through ligand-independent activation of LRP6/β-catenin signaling. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-022-02323-3.

with 5% CO2, and the medium was changed twice a week. After incubation, colonies were stained with the MTT solution (final 250-500 μg/mL). Colonies were imaged and counted using ImageJ software.

Real-time PCR
Total RNA was prepared using an easy-BLUE total RNA extraction kit (Intron Biotechnology, Sungnam-si, Kyunggi-do, Republic of Korea) according to the manufacturer's recommended procedure. We used a SYBR Green-based qPCR master mix solution (Enzynomics, Daejeon, Republic of Korea) and gene-specific primers. All real-time PCR assays were performed on an Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific). The following thermocycler conditions for real-time PCR were applied: pre-incubation at 95°C for 15 min; 40-70 cycles of 95°C for 10 sec, 60°C for 15 sec, and 72°C for 30 sec; and a final melt curve analysis to determine reaction specificity. Relative quantification of mRNA expression was performed using the comparative CT (cycle threshold) method as described in a previous report [1]. The primer sequences used in the PCR assays are shown in Table S3.

Plasmids, shRNAs, and Transfection
The plasmid constructs for hNinj1 expression are described in our previous report [2]. The plasmid construct for the FLAG-tagged hNinj1 protein was generated by using pCS+GFP hNinj1 vector as a template and subcloning them into the pCMV-Tag2B vector using the following primers: FLAG For GST-tagged bacterial protein expression, pCMV-Tag2B-hNinj1 was subcloned into the pGEX-4T-2. NT Ninj1 mutant was generated using the template pGEX-4T-2-FL-hNinj1 with the following primers: NT GST Ninj1 forward: 5'-CAGGAATTCCAATGGACTCGGGAAC-3', reverse: His-tagged bacterial protein expression, LRP6 and FZD2 domain constructs were subcloned into the pET32a. LRP6 constructs were generated by using LRP6-pCS2 vector as a template and FZD2 construct was generated by using pCMV6-AC-GFP h. Transfected cells were selected using G418.

Immunoprecipitation and pulldown assay
For immunoprecipitation analysis, cells were washed with ice-cold PBS twice and then harvested by IP lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM MgCl2, 10% glycerol, 100 mM NaF, 5 mM Na3VO4, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin] for 10 min on ice. After centrifugation at 13,000 rpm for 10 min at 4°C, supernatants were harvested, and protein concentration was determined by the BCA assay. 1 mg of protein was immunoprecipitated with primary antibodies overnight at 4°C in lysis buffer. Protein G agarose beads were added and incubated for additional 2 h. The beads were collected by centrifugation (3,000 rpm for 2 min at 4°C) and washed six times (three times with lysis buffer and three times with PBS). Bound proteins were extracted by boiling with 5x SDS-PAGE sample buffer for 5 min at 95°C. Proteins were resolved by SDS-PAGE, transferred onto PVDF membranes, and then subjected to Western blot analysis as described above.

Limiting dilution assay
Cells were harvested by trypsinization. Live cells, as confirmed using a trypan blue exclusion assay, were diluted in PBS, mixed with Matrigel (ratio 1:1), and then inoculated into the right flanks of NOD/SCID mice. The incidence of tumor formation was determined. Tumor-initiation fraction of vehicle-or Evo-treated groups was determined by using Extreme Limiting Dilution Analysis (ELDA) online software (http://bioinf.wehi.edu.au/software/elda/) [4].

Hanging drop assay
Cells (suspended in a complete medium) were seeded in 50 μL drops on the inner surface of a multi-well plate lid and cultured for a day. The drops were observed under an inverted microscope, and the non-aggregated cells were manually counted. Cells in at least four fields per sample were counted.

Cell adhesion assay
A 96-well culture plate was coated with type I collagen (10 μg/mL) and fibronectin (10 μg/mL)      The Ninj1 + cells were determined using ImageJ software. Scale bars: 20 μm. The significance of the correlation was determined using the Pearson correlation test. Statistical significance of the difference between two groups was determined by a two-tailed Student's t-test or Mann-Whitney test. Ninj1: Ninjurin1. TM: tamoxifen.